Activating film for chemiluminescent assays and methods for use

ABSTRACT

The present invention relates to chemiluminescent assays which incorporate a second film or membrane which includes a solid chemical component for activation of a stable dioxetane. Decomposition of the stable dioxetane can be accomplished using a combination of heat and chemical treatment.

This application is a national stage application of PCT/US00/03863,filed Feb. 16, 2000, which claimed benefit of provisional applicationSerial No. 60/120,125, filed Feb. 16, 1999.

FIELD OF THE INVENTION

The present invention relates to chemiluminescent assays whichincorporate a second film or membrane which includes a solid chemicalcomponent for activation of a stable dioxetane. Decomposition ofdioxetane can be accomplished using heat and chemical treatment.

BACKGROUND OF RELATED TECHNOLOGY

Recently a variety of non-isotopic labeling methods have been developedto replace radioactive labels in DNA probe-based assays. It is mostcommon in such methods to use marker enzymes to detect nucleic acidprobes using either colormetric, chemiluminescent, bioluminescent orfluorescent methods. Each of these methods have been used reliably forboth hybridization of DNA probe-based assays for nucleic acid detectionas well as in solid-phase immunochemical assays wherein the targetmolecule is typically an antigen of interest.

Regardless of the type of non-isotopic detection method used, the labelsare measured directly with fluorophores (without use of enzymes) orindirectly using enzyme amplification schemes. Wherein the label isdetected directly without an enzymatic reaction, sensitivity isgenerally less. Typically, in an indirect labeling scheme, a label isincorporated into the probe or the analyte in the form of a smallmolecule such as digoxigenin, fluorescein or biotin. This label may ormay not be detectable on its own and its presence is revealed usingenzyme conjugates that specifically bind to the small molecule in theprobe. A clear advantage of an indirect labeling scheme is the increasedsensitivity one achieves through enzymatic amplification of the signal.However, a disadvantage of such methods as they are currently practicedin the field is that many steps are required in the assay protocol,requiring more time to complete the assay. Moreover, a greater number ofreagents are required which means greater cost. In addition, where themethod of detection is enzyme-based, stability of the enzyme and itsshelf life need to be considered if one is to expect optimum performanceof the assay.

Chemiluminescence detection relies on a chemical reaction that generateslight. It is this method which is now widely used for both nucleic aciddetection as well as solid-based immunodetection due to its highsensitivity and wide variety of analysis methods ranging from manualfilm reading to instrumentation for processing images. Onenon-radioactive detection method now commercially available is theDIG-system (Boehringer-Mannheim) which uses digoxigenin, a smallmolecule, as a label for a probe. After binding of a DIG-labeled probeto a target molecule, an anti-DIG antibody conjugated to the enzymealkaline phosphatase is added. Detection is achieved through theenzymatic dephosphorylation of a 1,2-dioxetane substrate which leads tothe production of a chemiluminescent signal. This method relies on anenzymatic means of amplification of the signal and as such presents adisadvantage in that considerations regarding the stability of theenzyme and its shelf life are important. In addition, several steps arerequired in the protocol, including the binding of a probe to anenzyme-conjugated antibody. The shelf life of an antibody is anadditional consideration.

In view of the simplicity of chemical reactions relative to enzymaticreactions, it would be desirable to achieve chemiluminescent signalamplification by chemical as opposed to enzymatic means. U.S. Pat. No.5,516,636 to McCapra and a later publication by Schubert (Nucleic AcidsResearch, 1995, Vol. 23, No. 22 p. 4657) describe the use ofsensitizer-labeled oligonucleotide probes for the detection of nucleicacid target molecules. In a solid phase DNA probe assay, a DNA targetmolecule is bound to a membrane and hybridized to a sensitizer-labeledoligonucleotide complementary in sequence to the target DNA. Themembrane is subsequently treated with an olefin solution. Upon exposureof the membrane to ambient oxygen and light, the sensitizer moleculesbecome excited and transfer their excess energy to ambient oxygen forformation of singlet oxygen. The singlet oxygen therein produced reactswith the olefin on the membrane to form a stable 1,2-dioxetane in thearea of the hybridization zone which when subsequently exposed to heat,chemical treatment or enzymatic treatment decomposes to emit light.Thus, oligonucleotides labeled with sensitizer are able to amplify thedioxetane concentration based on repeated excitation/oxygen quenchingcycles to achieve a high level of sensitivity.

McCapra discloses that wherein the dioxetane contains a phenolichydroxyl protecting group the triggering chemical mechanism fordecomposition is the raising of pH. However, he fails to discloseparticular bases suitable for the decomposition or the exact method bywhich it can be accomplished.

Schubert uses chemical treatment of a thermally stable dioxetane as ameans of decomposing the dioxetane wherein decomposition is achieved bya change of pH using a liquid triggering solution of tetrabutyl ammoniumhydroxide. While an advantage of chemical treatment includes speed andefficiency, a disadvantage of the method of Schubert lies in the use ofa liquid base solution which can be caustic, inconvenient and messy touse. Schubert discloses that deprotonation via base treatment of thephenolic hydroxyl protecting group of the dioxetane used causes it tolose its thermal stability and decay with accompanying emission oflight.

The prior art fails to teach the combined use of heat and chemicaltreatment as a means of decomposing dioxetane. It would seem thereforethat there is a need for a method that could allow for the combined useof heat and chemical treatment as the triggering means for an evengreater enhancement of decomposition of a stable dioxetane. Thus, ifdecomposition of the dioxetane acts as the bottleneck for production ofa signal, it is important that the conditions under which decompositionoccur be optimal. Combining chemical treatment with heat would allow forthis. However, up until now it has not been possible to use bothchemical treatment and heat. Heating a caustic solution of base to at ornear boiling temperatures would be both dangerous and impractical.

There is therefore a need for a method of providing a chemicaltriggering agent in a dry form which upon exposure to an appropriateenergy source such as heat can be activated for enhanced decompositionof a thermally stable dioxetane and a resultant enhancement of thechemiluminescent signal. It would be a further advantage to provide fora way to use a similar dry agent to trigger dioxetane decomposition foruse in both solid-phase immunoassays and nucleic acid assays.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing preparation of the first film ofthe present invention used for nucleic acid chemiluminescent detection.

FIG. 2 is a schematic diagram showing preparation of the first film ofthe present invention used for antigen chemiluminescent detection.

FIG. 3 is a schematic diagram showing the conversion of an olefin, usedfor coating the film on which the target molecule is bound, forformation of a stable 1,2-dioxetane chemiluminescent precursor.

FIG. 4 is a perspective of a sandwich format of an assay of the presentinvention.

FIG. 5 is a perspective view of a sandwich assay format showingapplication of an electrical energy source to the second film of thepresent invention.

SUMMARY OF THE INVENTION

The present invention overcomes the disadvantages of currently practicedmethods for chemical decomposition of dioxetanes discussed above. Thepresent invention fulfills the need for a method by which a chemicaltriggering agent and heat can be combined for enhanced decomposition ofa thermally stable dioxetane and a resultant enhancement of achemiluminescent signal.

In one aspect of the present invention there is provided a film for usein chemiluminescent assays which includes a solid chemical componentimmobilized thereon or impregnated therein. The solid chemical componentwhen acted upon by an energy source releases an activating substancewhich in the presence of a chemiluminescent precursor compound reactstherewith to produce a chemiluminescent signal for the detection of atarget molecule. This film is used in the inventive assays along withanother film having the target molecule and sensitizer/probe hybridizedthereto.

In another aspect of the invention there is provided a chemiluminescentassay which includes a first and a second solid chemical componentimmobilized on or impregnated within film which when acted upon by anenergy source together allow for release of an activating substance at apH sufficient to produce a chemiluminescent signal upon reaction of saidactivating substance with a chemiluminescent precursor compound.Moreover, the invention involves specific binding chemiluminescentassays for the detection of target molecules wherein the targetmolecules can be nucleic acid or protein molecules.

In the assay, a probe is provided having a sensitizer as a label. Theprobe is capable of specifically binding to a target molecule in asample undergoing the assay. Following binding of the target moleculesto a first film, detection is carried out via hybridization with asensitizer labeled probe to form a solid film-bound complex. The firstsolid film-bound complex is separated from unbound probe and exposed toan olefin reagent. Subsequent exposure of the first film to light of aspecific wavelength range promotes the sensitizer to an excited statewhere it can transfer its excess energy to ambient molecular oxygen,with resultant formation of singlet oxygen. The singlet oxygen reactswith the olefin reagent to form a stable chemiluminescent precursorcompound. The method of using the inventive assay includes the steps ofcontacting a first film, on which is formed a stable chemiluminescentprecursor compound, with a second film having a solid chemical componentor components immobilized or impregnated therein; and exposing saidsecond film to an energy source which results in the release from thesecond film of an activating substance, which activating substance inthe presence of the stable chemiluminescent precursor compound on thefirst film reacts to produce a chemiluminescent signal for the detectionof target molecules.

A method of preparing the specific binding chemiluminescent assay of thepresent invention is described in the present invention. This methodincludes the steps of: (1) providing a first film having bound thereontarget molecules, wherein said bound target molecules have beensubjected to a pre-hybridization or blocking buffer solution undertemperatures suitable for a chosen probe and wherein said film isfurther incubated with hybridization solution containing said probeunder conditions suitable to bind said probe and wherein said film issubsequently exposed to an olefin solution; (2) Providing a second filmby immobilizing on or impregnating therein at least one solid chemicalcomponent which when acted upon by an energy source releases anactivating substance and further which in the presence of achemiluminescent precursor compound reacts therewith to producechemiluminescent signal for the detection of a target molecule; (3)positioning said first and second films in overlapping contact with eachother to permit release of an activating substance from said second filmand reaction with said chemiluminescent precursor compound on said firstfilm to result in a detectable chemiluminescent signal.

Moreover, a kit for performing the specific binding chemiluminescentassay for use in both nucleic acid binding assays as well asimmuno-assays is described. This kit includes: (1) a first film forbinding of a target molecule thereon; (2) a second film comprising atleast one solid chemical component immobilized on or impregnated withinsaid film, which chemical component when acted upon by an energy sourcereleases an activating substance which in the presence of achemiluminescent precursor compound reacts therewith to produce achemiluminescent signal for the detection of a target molecule.

A method of detecting target molecules using chemiluminescence is alsoprovided and includes the steps of: (a) providing a first film having acomplex comprising a target molecule bound to a sensitizer labeledprobe; (b) providing a second film comprising at least one solidchemical component immobilized on or impregnated on said film, whichchemical component when acted upon by an energy source releases anactivating substance which in the presence of a chemiluminescentprecursor compound reacts therewith to produce a chemiluminescent signalfor the detection of a target molecule; (c) contacting said complex withan olefin reagent to place said complex and said olefin reagent inreactive proximity to each other; (d) exposing said complex to light andoxygen to create a chemiluminescent precursor compound; (e) contactingsaid first film with said second film and allowing said second film tobe acted upon by an energy source to allow release from said second filmof an activating substance which in the presence of saidchemiluminescent precursor compound reacts to produce a chemiluminescentsignal for the detection of said target molecules; and (f) detectingand/or recording said resultant chemiluminescent signal.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect of the invention there is included a film for use in thedetection of target molecules via chemiluminescent solid-phase andgel-type assays. The film includes a solid chemical componentimmobilized on or impregnated within the phase which when acted upon byan energy source releases an activating substance, which substance inthe presence of a chemiluminescent precursor compound reacts therewithto produce a chemiluminescent signal for the detection of a targetmolecule.

The target molecule may be a nucleic acid, such as RNA or DNA. Inaddition, the film and its method of use may be used in solid-phaseimmunoassays in which the target molecule can be either antibody orantigen and wherein the corresponding sensitizer-labeled probe may beantigen or antibody, respectively. In the context of this invention, useof the term “film” includes membranes, filter paper and gels. Films maybe of any useful thickness or porosity depending on their specificapplication. Such films are inclusive of but not limited to textilefilms, paper films, cellulose films, polyacrylamide and agarose gels. Inparticular, it is envisioned that nylon, nitrocellulose, or PVDFmembranes or filter paper may be used for the release and transfer ofthe activating substance necessary for the chemiluminescent reaction tooccur.

Useful solid chemical components of the present invention include acids,bases, salts, enzymes, inorganic or organic catalysts, or electron donorsources. The specific solid chemical component chosen depends largely onthe identity of the chemical leaving group X shown in FIGS. 1-3.Referring now to FIG. 1, this schematic depicts a nucleic acidchemiluminescent assay wherein a target DNA molecule is bound to a film.A sensitizer-labeled oligonucleotide probe complimentary in sequence tothe target DNA is hybridized to the target DNA. Coating of the film withan olefin reagent, such as that shown in FIG. 3, is then performed.Subsequent to the exposure of the thus coated film to light and oxygen,the sensitizer is promoted to an excited state which allows for transferof its excess energy to ambient oxygen, with the resultant formation ofsinglet oxygen. Singlet oxygen reacts with the olefin reagent to form astable chemiluminescent 1,2-dioxetane, as shown in FIGS. 1-3. When thisfilm is placed in intimate contact with a second film of the presentinvention, as shown in FIGS. 4 and 5, an activating substance from thesolid chemical component on the second film is released by exposure toan energy source for reaction with the stable dioxetane on the firstfilm. Decomposition of the dioxetane subsequently occurs, with theresult being a detectable chemiluminescent signal.

Olefins having the structure shown in FIG. 3 have been described in U.S.Pat. No. 5,386,017 to Schaap. Treatment of a stable dioxetane located ona first film with the appropriate activating agent released from asecond film of the present invention produces chemiluminesence. The Xgroup on the dioxetane represents a labile leaving group. This group is“activated” or chemically cleaved by the solid chemical component on thesecond film. Examples of typical X groups which can be removedchemically as well as enzymatically are shown in U.S. Pat. No.5,795,987. Useful X-oxy protecting groups include, but are not limitedto, hydroxyl, alkyl or aryl carboxyl ester, inorganic oxy acid salt,alkyl or aryl silyloxy and oxygen pyranocide. Additional examples ofprotecting groups as well as the corresponding cleavage/activatingagents used for removal of X can also be found in the standard treatiseon protecting groups (reference Greene and Vuts, in Protective Groups inOrganic Synthesis, 1999).

Table 1 shows several activatable oxide groups (OX), which act asprotecting groups on the dioxetane. Table 1 also shows the correspondingsolid chemical component or components on the second film of the presentinvention, which are capable of removing the labile X group of thestable dioxetane to form the signal. The choice of solid chemicalcomponent will depend largely on the X group on the dioxetane. In thecase of X being hydrogen, deprotonation will be required in order todecompose the dioxetane for signal formation. In such as case, the solidchemical component when exposed to the proper energy source will releasea base. For example, the solid chemical component on the second film maybe ammonium carbonate, which when exposed to heat liberates a gaseousbase (NH₃), water, carbon dioxide and, if NH₃ reacts with water, hydroxy(OH—) anions. The basic components released then act to deprotonate thedioxetane resulting in signal formation.

The soluble chemical component or components on the second film of thepresent invention may be deposited thereon by such methods as coating,dipping, spraying, precipitating out of solution, and the like.

When the solid chemical component on the second film is an acid,desirable acids include but are not limited to benzoic acid andp-nitrobenzoic acid. When the solid chemical component on the film is abase, desirable bases include but are not limited to ammonium carbonate,ammonium carbamate, dibasic ammonium phosphate and potassium hydroxide.Useful salts for the solid chemical component include but are notlimited to sodium iodide, sodium metaphosphate trihexahydrate and sodiumorthophosphate-mono-H dodecahydrate. Where the solid chemical componentis a salt, it is desirable that it have a low melting temperature suchthat exposure to mild heat, e.g., 100° C., causes the salt to be meltedand hence, activated and made available for cleavage of the labilegroups on the stable dioxetane. In addition, bases released in thismanner, when exposed to mild heat conditions may combine with watervapor produced during heating and thereby become activated and availablefor cleavage of the labile group. Where the solid component is anenzyme, desired enzymes include alkaline phosphatase and horseradishperoxidase. The desirable means of activation of the enzyme would behydration in the presence of buffering salts.

TABLE 1 CORRESPONDING SOLID PROTECTING GROUP (OX) on CHEMICAL COMPONENTFOR DIOXETANE CLEAVING/PROTECTING GROUP Methoxymethyl ether NaI, BenzoicAcid 2,2 dichloro-1,1-difluoroethyl ether KOH trimethylsilyl ethertetrabutylammonium fluoride hydrate hydroxyl ammonium carbonate acetateester sodium carbonate, decahydrate phosphate alkaline phosphatase,Tris-Cl

As previously mentioned, the second film includes a solid chemicalcomponent which component may be selected from acids, bases, salts,enzymes, inorganic and organic catalysts, and electron donor sources andwhich is acted upon by an energy source to cause release of anactivating substance for production of a chemiluminescent signal on thefirst target molecule-bound film. This energy source may be chosen fromthermal energy, electromagnetic energy, electrical energy, mechanicalenergy, and combinations thereof. In the case where the solid chemicalcomponent is an enzyme, the energy source will be hydration, i.e.,exposure to sufficient water to release the buffered enzyme. Oneembodiment uses thermal energy as the energy source, wherein the energycomprises a temperature of about 30° to 100° C. Where the solid chemicalcomponent is a base in salt form with a melting temperature of 100° C.or lower, an application of a temperature of 100° C. or less causes thebase to melt and become activated to allow for reactivity with achemiluminescent precursor compound on another film for production witha chemiluminescent signal. Moreover, an increase in temperature resultsin the production of water and/or water vapor capable of dissolving abase or other solid component regardless of the melting temperature ofthe solid component and thereby activating it and making it availablefor reactivity with a chemiluminescent precursor compound on anotherfilm for signal production. Furthermore, production of water vapor uponheating causes formation of a gaseous base in certain instances whichcan become available for reactivity with a chemiluminescent precursor onanother film by diffusion. For example, wherein the base used isammonium carbonate, heating produces the gaseous base NH₃.

In yet another embodiment, an electromagnetic energy source comprised oflight having a wavelength about 30 nm to 1,100 nm is envisioned to beuseful in the generating of an activating substance on the solid phaseof the present invention. An additional energy source embodied by thepresent invention includes positioning the second film of the inventionwith another film (e.g., the first film) having a target moleculethereon to create intimate contact therebetween. If necessary, pressurecan be applied to create such intimate contact. Such intimate contactallows for diffusion of an activating substance from the second film ofthe present invention to the first film having a target molecule thereonto allow for production of a chemiluminescent signal.

In one desirable embodiment, the chemiluminescent precursor compound,which reacts with the activating substance released from the second filmof the present invention is a stable 1,2-dioxetane, the structure ofwhich is shown in FIG. 3. The preferred 1,2-dioxetane contains a labilechemical group X removable by enzymatic or chemical cleavage by theactivating substance, i.e., that which is released from the secondchemical component. Such dioxetanes are thermally stable and are knownin the art.

In another embodiment, the film of the present invention may furthercomprise a second chemical component, immobilized on or impregnatedwithin said phase which component when exposed to an energy sourcemaintains the pH of the activating substance within a range sufficientto produce a chemiluminescent signal. In this embodiment, the secondsolid chemical component may be an acid, base, salt or a combinationthereof. For example, wherein the labile group on the dioxetane requiresdeprotonation by a base, the first solid chemical component on the filmof the invention may be the salt ammonium bicarbonate, which whenexposed to mild heat, e.g., 80° C., forms water and/or water vapor, withconcomitant production of ammonia, carbon dioxide and OH anion. Carbondioxide, however, is sufficiently acidic to neutralize the basicenvironment, making deprotonation of the dioxetane less than optimal. Itmay be desirable, therefore, to include a second chemical component,such as potassium hydroxide, on the film to neutralize the acidic carbondioxide, such that the pH of the chemical environment is within a rangeto support optimal cleavage, i.e., about pH 13-14 of the labile group onthe stable dioxetane for resultant formation of a signal.

However, it should be noted that even in the absence of a secondchemical component such as potassium hydroxide, a high sensitivity ofdetection was achieved with ammonium bicarbonate as the sole solidchemical component, combined with mild heat with as few as 25 fmoles oftarget DNA detectable.

When the second solid chemical component is an acid, desirable acidsinclude, but are not limited to, benzoic acid and p-nitrobenzoic acid.When the second solid chemical component is a base, desirable basesinclude, but are not limited to, sodium hydroxide, potassium hydroxideand sodium carbonate decahydrate. A useful salt for the second solidchemical component includes, but is not limited to sodium sulfatedecahydrate.

In one embodiment, the second chemical component is or releases a basewherein the pH is maintained at equal to or about 8. In a preferredembodiment, the second solid chemical component is a base, or a saltwhich releases a base which together with the first solid chemicalcomponent, maintains the pH at about 13 to about 14. The usefulness ofthis invention is meant to extend to solid-phase immunoassays, as wellas nucleic acid assays. Although exposure of an antibody-antigen complexto high pH would normally not be desirable, it is noted that even if theantibody-antigen complex were to dissociate, the ability to detect asignal would not be adversely effected. This is because followingexposure of the olefin coated membrane to light and oxygen (air),singlet oxygen is formed which allows for conversion of the olefin to astable dioxetane, but only in the hybridization zone. Thus, subsequentexposure to base with resulting deprotonation of the dioxetane willyield a signal regardless of whether the antibody-antigen complexremains intact.

Alternatively, wherein the solid chemical component required foractivation of the stable dioxetane is an enzyme, the second solidchemical component may be a buffering salt, including but not limited toTris.Cl which is useful for maintaining the pH within a neutral range asrequired for maximum catalytic activity of most enzymes.

Referring now to FIG. 4, there is a depiction of a sandwich formationuseful for the proper positioning of the two films. Film 4 represents afilm having a hybridized target molecule and triggerable dioxetanethereon. Film 5 represents the second film of the present inventionwhich contains the solid chemical component or components. Films 4 and 5are positioned in overlapping relationship and in intimate contacttherebetween. A transparent protective film 3 is positioned betweenphotographic film 2 and film 4. The entire sandwich structure issupported by glass plates 1 and 6, respectively. Energy source 7 isapplied to the sandwich format of FIG. 5 to activate and/or release thechemical component needed to react with the stable dioxetane in film 4.As noted above, various types of energy sources are useful. Positioningof photographic film 2 in this manner allows for capture of thechemiluminescent signal. It should be noted, however, that other meansof signal detection and capture may be utilized in place of thephotographic film. Electronic devices may be useful in this regard. FIG.5/5 shows application of a voltage to second film 5. This application ofelectrical energy may serve to apply heat to or cause ion flow in thesolid chemical component on film 5.

The following non-limited examples are provided but are not intended tolimit the scope or spirit of the invention in any way.

EXAMPLES Example 1 Sensitizer Labeling of Oligonucleotide

Modified methylene blue derivatives were obtained according toprocedures described by Motsenbocker, et al. The terminal carboxy groupof an activated N-hydroxysuccinimido ester form of the methylene bluesensitizer was coupled to a 5′-aminomodified oligonucleotide usingstandard methods known in the art. (Ruth, J. L., in Oligonucleotides andAnalogues: A Practical Approach, Eckstein (Editor), pp. 255-280, OxfordUniversity Press, NY 1991). The oligonucleotide was complementary insequence to cDNA encoding alcohol dehydrogenase. The 5′-aminomodifiedoligonucleotides used for labeling with methylene blue as well asunmodified oligonucleotides used for PCR amplification of the targetalcohol dehydrogenase cDNA were synthesized on a PE Biosystems NucleicAcid Synthesizer, Model No. ABI 3948.

Example 2 Dot Blot Hybridization of Methylene Blue-labeledOligonucleotide to Target DNA

Following PCR amplification of the target DNA, said DNA was spotted on aHybond+ nylon membrane (Amersham-Pharmacia), along with negativecontrols of linearized pUC19 DNA at various concentrations ranging from25 to 500 fmoles in a total volume of 1 microliter. Spots were allowedto dry. The DNA was subsequently denatured and fixed as follows: 1minute soak in 1.5 M NaCl; 0.5 M NaOH, followed by fixation by baking at120° C. for 40 minutes, followed by 5 minute soak in 1.5 M NaCl; 0.5 MTris-Cl pH 7.5. Hybridization was as follows: The filter membrane wassoaked in prehybridization buffer (0.25 M Na—PO₄; pH 7.2; 7% (w/v) SDSfor 45 minutes at 40° C. in a total volume of 0.5 ml per cm² membrane.The labeled oligonucleotide probe was added directly to theprehybridization buffer at a final concentration of about 2 umoles/mland incubated for 16 h at 40° C. The hybridized membrane was washed in abuffer of 6×SSC at room temperature for two times at 5 minutes each washfollowed by two times at 5 minutes each wash in 3×SSC at 40° C. toremove nonstringent or background hybridization.

Example 3 Method of Detecting Target DNA Hybridized to MethyleneBlue-labeled Oligonucleotide

Hybridization was detected by first briefly (less than 5 seconds)dipping the hybridized membrane in an olefin solution (1/100% w/v inhexane or methanol), wherein olefin was synthesized by the method ofSchaap as described in U.S. Pat. No. 4,857,652, and allowing it to airdry, then illuminating the hybridized surface with red light for 15minutes. In order to detect the signal, a sheet of filter paperpreviously soaked in a saturated solution of ammonium carbonate and thendried to a solid form was taped to a glass plate. The hybridizedmembrane with bound target DNA was subsequently placed (DNA side up) ontop of the filter paper containing the dried base and a sheet of plasticwas placed on top of this. In the dark, a sheet of Hyperfilm ECL(Amersham-Pharmacia) was placed over the plastic sheet and another glassplate was placed on top. The whole sandwich formation was incubated at80° C. for 15 minutes to allow for release of the base from the filterpaper and resultant activation of the stable chemiluminescent precursorcompound present on the hybridized membrane. The film was developedusing standard techniques and successful hybridization was observed asblack spots on the Hyperfilm ECL with the lowest quantity of DNAdetected being in the range of 25 fmoles.

Example 4 Sensitizer Labeling of an Antibody or Antigen

A modified methylene blue derivative is obtained according to proceduresdescribed by Motsenbocker, et al. The terminal carboxy group of anactivated N-hydroxysuccinimido ester form of the methylene bluesensitizer is coupled to an antibody or antigen via the terminal aminogroup using standard methods known in the art. The antibody probe usedis specific for an antigen target molecule or alternatively, an antigenprobe is specific for an antibody target molecule.

Example 5 Dot Blot Hybridization of Methyleneblue-labeled Antibody Probeto Target Antigen Molecules

Antigen is spotted on a nitrocellulose, PVDF or nylon membrane invarious concentrations. The membrane is subsequently blocked in asolution of 0.2% casein/0.1% Tween 20 detergent in aqueous phosphatebuffered saline solution (PBS) for 1 hour, following which a 1/2000 to1/5000 dilution of methylene blue-labeled antibody in 0.2% casein in PBSis added, wherein the antibody is specific for the target antigenmolecule. The membrane is then incubated for 1 hour at room temperatureand washed five times (for 5 minutes each time) in 0.3% Tween 20detergent in PBS, and one time in PBS at room temperature for 5 minutesto remove non-specific or non-stringent binding.

Example 6 Method of Detecting Target Antigen Hybridized to MethyleneBlue-labeled Antibody

Hybridization is detected by first briefly (less than 5 seconds) dippingthe hybridized membrane in an olefin solution (1/100% w/v in hexane ormethanol and allowing it to air dry, then illuminating the hybridizedsurface with red light for 15 minutes. In order to detect the signal, asheet of filter paper previously soaked in a saturated solution ofammonium carbonate and then dried to a solid form is taped to a glassplate. The hybridized membrane with bound target antigen is subsequentlyplaced (antigen side up) on top of the filter paper containing the driedbase and a sheet of plastic is placed on top of this. In the dark, asheet of Hyperfilm ECL (Amersham-Pharmacia) is placed over the plasticsheet and another glass plate is placed on top. The whole sandwichformation is incubated at 80° C. for 15 minutes to allow for release ofthe base from the filter paper and resultant activation of the stablechemiluminescent precursor compound present on the hybridized membrane.The film is developed using standard techniques for signal detection.

What is claimed is:
 1. A film for use in chemiluminescent assayscomprising at least one solid chemical component immobilized on orimpregnated therewith which when acted upon by an energy source releasesan activating substance, which substance in the presence of achemiluminescent precursor compound reacts therewith to produce achemiluminescent signal for the detection of a target molecule.
 2. Afilm according to claim 1 wherein said film comprises a polymeric film.3. A film according to claim 1 wherein said film further comprises atextile, paper or cellulose film.
 4. A film according to claim 1 whereinsaid solid chemical component is selected from the group consisting ofacids, bases, salts, enzymes, inorganic and organic catalysts, andelectron donor sources.
 5. A film according to claim 1 wherein saidsolid chemical component is an acid selected from the group consistingof benzoic acid and p-nitrobenzoic acid.
 6. A film according to claim 1wherein said solid chemical component is a base selected from the groupconsisting of ammonium carbonate, ammonium carbamate, dibasic ammoniumphosphate and potassium hydroxide.
 7. A film according to claim 1wherein said solid chemical component is a salt selected from the groupconsisting of sodium iodide, sodium metaphosphate trihexahydrate, sodiumorthophosphate-mono-H dodecahydrate.
 8. A film according to claim 1wherein said energy source is selected from the group consisting ofthermal energy, electromagnetic energy, electrical energy, mechanicalenergy and combinations thereof.
 9. A film according to claim 1 whereinsaid energy source comprises a temperature of about 30° to about 100° C.10. A film according to claim 1 wherein said electromagnetic energysource comprises light having a wavelength from about 30 nm to about1,100 nm.
 11. A film according to claim 1 wherein said mechanical energycomprises application of pressure to said film to create intimatecontact with another film having a target molecule thereon.
 12. A filmaccording to claim 1 wherein said chemiluminescent precursor compound isa 1,2-dioxetane.
 13. A film according to claim 12 wherein the1,2-dioxetane contains a labile chemical group removable by chemical orenzymatic cleavage by said activating substance.
 14. A film according toclaim 12 wherein the 1,2 dioxetane is formed from an olefin.
 15. A filmaccording to claim 14 wherein the olefin is covalently bound to afluorescent molecule which further enhances chemiluminescent detection.16. A film according to claim 1 wherein said film further comprises asecond solid chemical component immobilized on or impregnated withinsaid film which component when exposed to said energy source maintainsthe pH of said activating substance within a range sufficient to producea chemiluminescent signal.
 17. A film according to claim 16 wherein saidsecond solid chemical component is selected from the group consisting ofacids, bases, salts and combinations thereof.
 18. A film according toclaim 16 wherein said second solid chemical component is an acidselected from the group consisting of benzoic acid, p-nitrobenzoic acid.19. A film according to claim 16 wherein said second solid chemicalcomponent is a base selected from the group consisting of sodiumhydroxide, potassium hydroxide, sodium carbonate decahydrate.
 20. A filmaccording to claim 16 wherein said second chemical component is the saltsodium sulfate decahydrate.
 21. A film according to claim 16 whereinsaid second solid chemical component is or releases a base and whereinsaid pH is equal to about
 8. 22. A film according to claim 16 whereinsaid second solid chemical component is or releases a base and whereinsaid pH is 13-14.
 23. A film for use in the detection of a targetmolecule in solid-phase and gel chemiluminescent assays comprising firstand second solid chemical components immobilized on or impregnatedwithin a film or membrane which components when acted upon by an energysource together allow for release of an activating substance at a pHsufficient to produce a chemiluminescent signal upon reaction of saidactivating substance with a chemiluminescent precursor compound.
 24. Afilm for use in chemiluminescent assays comprising at least one solidchemical component immobilized on or impregnated therein which whenhydrated releases an activating substance, which substance in thepresence of a chemical precursor compound reacts therewith to produce achemiluminescent signal for the detection of a target molecule.
 25. Afilm according to claim 24 wherein said film comprises a polymeric film.26. A film according to claim 25 wherein said film comprises a textileor cellulose film.
 27. A film according to claim 24 wherein said solidchemical component is an enzyme.
 28. A film according to claim 24wherein said solid chemical component is an enzyme selected from thegroup consisting of alkaline phosphatase and horseradish peroxidase. 29.A film according to claim 24 wherein said chemiluminescent precursorcompound is a 1,2 dioxetane.
 30. A film according to claim 29 whereinsaid dioxetane contains a labile group removable by enzymatic cleavage.31. A film according to claim 24 wherein said film further comprises asecond solid chemical component immobilized on or impregnated withinsaid film which when exposed to hydration maintains the pH of saidactivating substance within a range sufficient to produce achemiluminescent signal.
 32. A film according to claim 23 wherein saidsecond solid chemical component is selected from the group consisting ofacids, bases, salts and combinations thereof.
 33. A film according toclaim 23 wherein said second solid chemical component is Tris.Cl.
 34. Amethod of detecting target molecules using chemiluminescence comprisingthe steps of: (a) providing a first film having a complex comprising atarget molecule bound to a sensitizer labeled probe; (b) providing asecond film comprising at least one solid chemical component immobilizedon or impregnated on said film, which chemical component when acted uponby an energy source releases an activating substance which in thepresence of a chemiluminescent precursor compound reacts therewith toproduce a chemiluminescent signal for the detection of a targetmolecule; (c) contacting said complex with an olefin reagent to placesaid complex and said olefin reagent in reactive proximity to eachother; (d) exposing said complex to light and oxygen to create achemiluminescent precursor compound; (e) contacting said first film withsaid second film and allowing said second film to be acted upon by anenergy source to allow release from said second film of an activatingsubstance which in the presence of said chemiluminescent precursorcompound reacts to produce a chemiluminescent signal for the detectionof said target molecules; and (f) detecting and/or recording saidresultant chemiluminescent signal.
 35. The method of claim 34 whereinthe second film is a polymeric, textile, paper or cellulose film. 36.The method of claim 34 wherein said solid chemical component is selectedfrom the group consisting of acids, bases, salts, enzymes inorganiccatalysts and organic catalysts.
 37. The method of claim 34 wherein saidsolid chemical component is benzoic acid or p-nitrobenzoic acid.
 38. Themethod of claim 34 wherein said chemical component is ammoniumcarbonate, ammonium carbamate, dibasic ammonium phosphate, potassiumhydroxide and combinations thereof.
 39. The method of claim 34 whereinsaid solid component is an enzyme selected from the group consisting ofalkaline phosphatase and horseradish peroxidase.
 40. The method of claim34 wherein said solid chemical component is an enzyme and said energysource is hydration.
 41. A chemiluminescent assay kit comprising: (1) afirst film for binding of a target molecule thereon; (2) a second filmcomprising at least one solid chemical component immobilized on orimpregnated within said film, which chemical component when acted uponby an energy source releases an activating substance which in thepresence of a chemiluminescent precursor compound reacts therewith toproduce a chemiluminescent signal for the detection of a targetmolecule.
 42. The assay kit of claim 41 further including a buffersolution suitable for hybridization of proteins.
 43. The assay kit ofclaim 41 further including a buffer solution for hybridization ofantibodies or antigens.
 44. The assay kit of claim 41 further includinga buffer solution for hybridization of nucleic acids.
 45. The assay kitof claim 41 further including a buffer solution suitable for blocking ofproteins.
 46. The assay kit of claim 41 further including a buffersolution suitable for pre-hybridization of nucleic acids.
 47. The assaykit of claim 41 further including a washing buffer solution forproteins.
 48. The assay kit of claim 41 further including a washingbuffer solution for nucleic acids.
 49. The assay kit of claim 41 whereinsaid solid chemical component is selected from the group consisting ofacids, bases, salts, enzymes, inorganic catalysts, organic catalysts andcombinations thereof.
 50. The assay kit of claim 41 wherein said solidchemical component is a base selected from the group consisting ofammonium carbonate, ammonium carbamate, dibasic ammonium phosphate,potassium hydroxide and combinations thereof.
 51. The assay kit of claim41 further including an activated ester form of a sensitizer forreaction with proteins and aminomodified oligonucleotides.
 52. Themethod of claim 41 wherein said solid chemical component is an enzymeand said energy source is hydration.
 53. A method of preparing achemiluminescent assay comprising the steps of: (1) providing a firstfilm having bound thereon target molecules, wherein said bound targetmolecules have been subjected to a pre-hybridization or blocking buffersolution under temperatures suitable for a chosen probe and wherein saidfilm is further incubated with hybridization solution containing saidprobe under conditions suitable to bind said probe and wherein said filmis exposed to an olefin solution; (2) providing a second film byimmobilizing on or impregnating therein at least one solid chemicalcomponent which when acted upon by an energy source releases anactivating substance and further which in the presence of achemiluminescent precursor compound reacts therewith to producechemiluminescent signal for the detection of a target molecule; (3)positioning said first and second films in overlapping contact with eachother to permit release of an activating substance from said second filmand reaction with said chemiluminescent precursor compound on said firstfilm to result in a detectable chemiluminescent signal.
 54. The methodof claim 53 wherein prior to positioning said first and second films inoverlapping relationship, said first film is exposed to sufficient lightand oxygen to form a stable chemiluminescent precursor compound.
 55. Themethod of claim 53 wherein said stable chemiluminescent precursorcompound is formed from the transfer of energy from said sensitizermolecules to ambient molecular oxygen with the resultant formation ofsinglet oxygen, said singlet oxygen subsequently reacting with saidolefin to form said stable chemiluminescent precursor compound.
 56. Themethod of claim 53 wherein said first and second films positioned inoverlapping relationship are placed proximate to a detection device forcapture of said chemiluminescent signal.
 57. The method of claim 53wherein said detection device is x-ray film or photographic film. 58.The method of claim 53 wherein said device is an electronic sensingdevice.